stically alter transfection results. Different cells or cell types have very specific medium, serum, and supplement requirements. We recommend using cells with a low passage number (<50 splitting cycles) to ensure that the cell genotype does not become altered.

Serum
In general, the use of serum enhances transfection. However, to avoid contamination with ribonucleases (RNases), we do not recommend using serum during transfection of RNA.

RNA structure
The structure of the transfected RNA molecule can influence the efficiency of transfection by influencing the formation of the TransMessenger ReagentRNA complex. In addition, the presence of specific RNA characteristics, such as a cap, poly-A tail, or internal ribosomal entry site (IRES), may increase or decrease the efficiency of translation in a given cell type. Messenger RNA (mRNA) species in eukaryotic cells usually terminate at the 5'-end with a cap and at the 3'-end with a poly-A tail. A cap is a modified nucleotide incorporated at the 5'-end of RNA transcripts that enhances mRNA stability and is necessary in some cell types to ensure optimal translation. A cap can be incorporated into an in vitro-transcribed RNA by adding an RNA cap-structure analog to the transcription reaction. A poly-A tail, a stretch of adenylate residues added 3' to the mRNA coding sequence, also stabilizes mRNA. A poly-A tail can be incorporated into an in vitro-transcribed RNA by using a relevant DNA template or added post-transcriptionally using a poly-A polymerase. An IRES is a specific sequence which helps the ribosome to access the mRNA, thereby enhancing translation. However in some cell types, RNA species containing an
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